"Classical" sexually transmitted infections (STIs) and bacterial vaginosis (BV) are associated with HIV susceptibility and infectivity. However, trials of STI control for HIV prevention have been largely negative. We hypothesize that other genital organisms may induce inflammation and contribute to increased risk of HIV. We will use data and samples collected during a trial of male circumcision (MC) to assess this hypothesis. HIV-negative men were randomized to immediate MC (intervention) or MC delayed by 2 years (controls). Men provided blood and penile swabs at 0, 6, 12 and 24 months. Foreskins were preserved at time of MC. Female partners of these men provided blood and vaginal swabs at 0, 12 and 24 months. Using these materials, we will address the following aims: Aim1. 1a) Assess the correlation between the genital burden of anaerobes and levels of genital cytokines/chemokines in 100 intervention and 100 control men and their 200 female partners.1.b) Assess the association between these exposures and histologic inflammation in foreskin tissues. 1.c. Using the data from Aim 1.a, we will assess the effects of MC on genital anaerobes and inflammatory markers in intervention versus control arm men and their partners. Aim 2. Assess the burden of genital anaerobes and levels of cytokines/chemokines associated with HIV acquisition using a case-control analysis of 105 male and 89 female HIV seroconverters, and 388 HIV-uninfected controls. Adjusted odds ratios will be estimated by multivariable logistic regression. Aim 3. In 310 HIV discordant couples we will estimate rates of HIV transmission/acquisition associated with genital anaerobe burden and cytokine/ chemokine levels. Incidence rate ratios of HIV acquisition/transmission will be estimated by Poisson multivariable regression. In an additional Aim 4, we will utilize the data from Aims 1-3 to assess the mediating role of anaerobes and inflammation on MC efficacy for HIV prevention in men. Laboratory methods: Total bacterial load, and the relative and absolute anaerobe burdens will be assessed by16S rRNA qPCR and 16SrRNA gene-based pyrosequencing. Cytokine and chemokine concentrations will be assayed using multiplex sandwich ELISA (SearchLight, Aushon Biosystems, MA) for TNFa, IFN?, IL-1[unreadable], IL-8, IL-6, IL-12, RANTES, Monokine Induced by IFN? (MIG/CXCL9), Macrophage Chemotactic Protein (MCP1/CCL2), and Interferon Inducible Protein-10 (IP10/CXCL10). This is a multidisciplinary collaboration between epidemiologists, molecular biologists and immunologists, and includes a new investigator (Dr. L Price, co-PI). PUBLIC HEALTH RELEVANCE: If genital anaerobes cause mucosal inflammation and are associated with HIV acquisition/transmission, the study could facilitate development of novel approaches to HIV prevention by control of pro-inflammatory genital anaerobes (e.g., by use of topical antiseptics or antibiotics), or agents to modulate genital inflammatory responses in men and women. This could inform microbicide development. Insights into mucosal immunology may also contribute to development of future mucosal HIV vaccines.